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A novel chromatin protein, distantly related to histone H2A, is largely excluded from the inactive X chromosome.

Chadwick BP, Willard HF - J. Cell Biol. (2001)

Bottom Line: In both interphase and metaphase female cells, using either a myc epitope-tagged or green fluorescent protein-tagged H2A-Bbd construct, the inactive X chromosome is markedly deficient in H2A-Bbd staining, while the active X and the autosomes stain throughout.In double-labeling experiments, antibodies to acetylated histone H4 show a pattern of staining indistinguishable from H2A-Bbd in interphase nuclei and on metaphase chromosomes.The distribution of H2A-Bbd thus distinguishes chromatin on the active and inactive X chromosomes.

View Article: PubMed Central - PubMed

Affiliation: Department of Genetics, Case Western Reserve University School of Medicine and Center for Human Genetics and Research Institute, University Hospitals of Cleveland, Cleveland, Ohio 44106-4955, USA.

ABSTRACT
Chromatin on the mammalian inactive X chromosome differs in a number of ways from that on the active X. One protein, macroH2A, whose amino terminus is closely related to histone H2A, is enriched on the heterochromatic inactive X chromosome in female cells. Here, we report the identification and localization of a novel and more distant histone variant, designated H2A-Bbd, that is only 48% identical to histone H2A. In both interphase and metaphase female cells, using either a myc epitope-tagged or green fluorescent protein-tagged H2A-Bbd construct, the inactive X chromosome is markedly deficient in H2A-Bbd staining, while the active X and the autosomes stain throughout. In double-labeling experiments, antibodies to acetylated histone H4 show a pattern of staining indistinguishable from H2A-Bbd in interphase nuclei and on metaphase chromosomes. Chromatin fractionation demonstrates association of H2A-Bbd with the histone proteins. Separation of micrococcal nuclease-digested chromatin by sucrose gradient ultracentrifugation shows cofractionation of H2A-Bbd with nucleosomes, supporting the idea that H2A-Bbd is incorporated into nucleosomes as a substitute for the core histone H2A. This finding, in combination with the overlap with acetylated forms of H4, raises the possibility that H2A-Bbd is enriched in nucleosomes associated with transcriptionally active regions of the genome. The distribution of H2A-Bbd thus distinguishes chromatin on the active and inactive X chromosomes.

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Chromosomal localization of H2A-Bbd on metaphase chromosomes from the female embryonic kidney carcinoma cell line 293. (a) Partial metaphase spread of a H2A-Bbd–transfected 293 cell showing the DAPI staining of the chromosomes (blue) merged with X alpha satellite cyanine 5.18 (Cy5) FISH signals (pink). Three X chromosomes are indicated with white arrows. (b) Indirect immunofluorescence of H2A-Bbd distribution on the same partial metaphase spread. Two of the X chromosomes do not stain with H2A-Bbd, while one (the cytologically marked Xa) shows a similar pattern to the surrounding autosomes (red, TR). The X alpha satellite Cy5 signal is shown (pink) and the positions of the X chromosomes are indicated with white arrows.
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Figure 3: Chromosomal localization of H2A-Bbd on metaphase chromosomes from the female embryonic kidney carcinoma cell line 293. (a) Partial metaphase spread of a H2A-Bbd–transfected 293 cell showing the DAPI staining of the chromosomes (blue) merged with X alpha satellite cyanine 5.18 (Cy5) FISH signals (pink). Three X chromosomes are indicated with white arrows. (b) Indirect immunofluorescence of H2A-Bbd distribution on the same partial metaphase spread. Two of the X chromosomes do not stain with H2A-Bbd, while one (the cytologically marked Xa) shows a similar pattern to the surrounding autosomes (red, TR). The X alpha satellite Cy5 signal is shown (pink) and the positions of the X chromosomes are indicated with white arrows.

Mentions: As shown above, H2A-Bbd clearly localizes to the nucleus of interphase cells and is largely excluded from the Xi chromosome in female cells. To investigate whether H2A-Bbd remains associated with metaphase chromosomes during mitosis, we examined the female embryonic kidney carcinoma cell line 293, which contains a single Xa (marked cytogenetically by a deletion of the short arm) and a variable number of Xi's (one to four copies in different cells). The 293 cells were transfected with myc-tagged H2A-Bbd and metaphase chromosomes were prepared. Immunolocalization followed by FISH to identify the X chromosomes revealed a distinct H2A-Bbd staining pattern. There was no obvious enrichment for any chromosomal regions; however, there was an almost complete absence of signal on the Xi (Fig. 3). Over 90% of metaphase spreads showed the same pattern; staining of the Xa was indistinguishable from autosomes, while all additional Xs stained poorly (Table ). A control experiment using a myc-tagged H2B control construct showed all X chromosomes and autosomes having an identical homogeneous staining (data not shown). As an additional control, metaphase spreads were made from the male tetraploid tumour line HT1080 transfected with H2A-Bbd. In the vast majority of spreads, the X chromosomes showed homogeneous H2A-Bbd staining (Table ).


A novel chromatin protein, distantly related to histone H2A, is largely excluded from the inactive X chromosome.

Chadwick BP, Willard HF - J. Cell Biol. (2001)

Chromosomal localization of H2A-Bbd on metaphase chromosomes from the female embryonic kidney carcinoma cell line 293. (a) Partial metaphase spread of a H2A-Bbd–transfected 293 cell showing the DAPI staining of the chromosomes (blue) merged with X alpha satellite cyanine 5.18 (Cy5) FISH signals (pink). Three X chromosomes are indicated with white arrows. (b) Indirect immunofluorescence of H2A-Bbd distribution on the same partial metaphase spread. Two of the X chromosomes do not stain with H2A-Bbd, while one (the cytologically marked Xa) shows a similar pattern to the surrounding autosomes (red, TR). The X alpha satellite Cy5 signal is shown (pink) and the positions of the X chromosomes are indicated with white arrows.
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Figure 3: Chromosomal localization of H2A-Bbd on metaphase chromosomes from the female embryonic kidney carcinoma cell line 293. (a) Partial metaphase spread of a H2A-Bbd–transfected 293 cell showing the DAPI staining of the chromosomes (blue) merged with X alpha satellite cyanine 5.18 (Cy5) FISH signals (pink). Three X chromosomes are indicated with white arrows. (b) Indirect immunofluorescence of H2A-Bbd distribution on the same partial metaphase spread. Two of the X chromosomes do not stain with H2A-Bbd, while one (the cytologically marked Xa) shows a similar pattern to the surrounding autosomes (red, TR). The X alpha satellite Cy5 signal is shown (pink) and the positions of the X chromosomes are indicated with white arrows.
Mentions: As shown above, H2A-Bbd clearly localizes to the nucleus of interphase cells and is largely excluded from the Xi chromosome in female cells. To investigate whether H2A-Bbd remains associated with metaphase chromosomes during mitosis, we examined the female embryonic kidney carcinoma cell line 293, which contains a single Xa (marked cytogenetically by a deletion of the short arm) and a variable number of Xi's (one to four copies in different cells). The 293 cells were transfected with myc-tagged H2A-Bbd and metaphase chromosomes were prepared. Immunolocalization followed by FISH to identify the X chromosomes revealed a distinct H2A-Bbd staining pattern. There was no obvious enrichment for any chromosomal regions; however, there was an almost complete absence of signal on the Xi (Fig. 3). Over 90% of metaphase spreads showed the same pattern; staining of the Xa was indistinguishable from autosomes, while all additional Xs stained poorly (Table ). A control experiment using a myc-tagged H2B control construct showed all X chromosomes and autosomes having an identical homogeneous staining (data not shown). As an additional control, metaphase spreads were made from the male tetraploid tumour line HT1080 transfected with H2A-Bbd. In the vast majority of spreads, the X chromosomes showed homogeneous H2A-Bbd staining (Table ).

Bottom Line: In both interphase and metaphase female cells, using either a myc epitope-tagged or green fluorescent protein-tagged H2A-Bbd construct, the inactive X chromosome is markedly deficient in H2A-Bbd staining, while the active X and the autosomes stain throughout.In double-labeling experiments, antibodies to acetylated histone H4 show a pattern of staining indistinguishable from H2A-Bbd in interphase nuclei and on metaphase chromosomes.The distribution of H2A-Bbd thus distinguishes chromatin on the active and inactive X chromosomes.

View Article: PubMed Central - PubMed

Affiliation: Department of Genetics, Case Western Reserve University School of Medicine and Center for Human Genetics and Research Institute, University Hospitals of Cleveland, Cleveland, Ohio 44106-4955, USA.

ABSTRACT
Chromatin on the mammalian inactive X chromosome differs in a number of ways from that on the active X. One protein, macroH2A, whose amino terminus is closely related to histone H2A, is enriched on the heterochromatic inactive X chromosome in female cells. Here, we report the identification and localization of a novel and more distant histone variant, designated H2A-Bbd, that is only 48% identical to histone H2A. In both interphase and metaphase female cells, using either a myc epitope-tagged or green fluorescent protein-tagged H2A-Bbd construct, the inactive X chromosome is markedly deficient in H2A-Bbd staining, while the active X and the autosomes stain throughout. In double-labeling experiments, antibodies to acetylated histone H4 show a pattern of staining indistinguishable from H2A-Bbd in interphase nuclei and on metaphase chromosomes. Chromatin fractionation demonstrates association of H2A-Bbd with the histone proteins. Separation of micrococcal nuclease-digested chromatin by sucrose gradient ultracentrifugation shows cofractionation of H2A-Bbd with nucleosomes, supporting the idea that H2A-Bbd is incorporated into nucleosomes as a substitute for the core histone H2A. This finding, in combination with the overlap with acetylated forms of H4, raises the possibility that H2A-Bbd is enriched in nucleosomes associated with transcriptionally active regions of the genome. The distribution of H2A-Bbd thus distinguishes chromatin on the active and inactive X chromosomes.

Show MeSH
Related in: MedlinePlus